Editor
Alexander Razdan, PhD
Associate Editors
John Osborn, BSc
Oki Dzivenu, DPhil
Jim Malone, MSc
Christina Ninalga, PhD
Production
Pär Jansson
Discovery Matters is published by
GE Healthcare.
The goal of Discovery Matters is to
provide you with information that
will help you achieve your research
objectives. We want to continue
developing Discovery Matters into
a publication you value, and
appreciate your input. Please send
your articles for submission in
upcoming issues, plus any
comments or questions to:
alex.razdan@ge.com

Nucleic acid purification
Comparative analysis of illustra triplePrep Kit for the isolation
of genomic DNA, total RNA, and total denatured protein from
a single undivided sample

16-17

GE & Science Prize for Young Scientists 2008
Understanding a minimal DNA-segregating machine Reconstitution of a plasmid spindle shows how three components
together accomplish the task of DNA segregation

18–19

To receive Discovery Matters,
subscribe online at:
www.gelifesciences.com
The content of this issue and
previous issues of Discovery
Matters can also be viewed online at
www.gelifesciences.com/
discoverymatters

Have you seen the Products for
Life Sciences 2008/2009 catalog?
The new catalog is a comprehensive guide
to the majority of products for life science
research from GE Healthcare. Order your
copy online at:
www.gelifesciences.com/catalog

>> Convenience: ready-to-use buffers save time by eliminating the
need for homemade buffers. The Yeast Protein Extraction Buffer Kit
allows for efficient extraction without having to use glass beads

Yeast Protein Extraction Buffer Kit

The 2-D Protein Extraction Buffer Trial Kit contains each of the six
extraction buffers, allowing you to screen for the most appropriate
extraction buffer for your sample. The extraction buffers can also be
used prior to 1-D electrophoresis, or for rehydrating IPG strips prior
to 2-D electrophoresis.
Key benefits include:

Efficient cell lysis and protein extraction are key steps for achieving
high quality results in downstream applications such as 2-D gel analysis.
The 2-D Protein Extraction Buffers offer a convenient way to prepare
high quality protein lysates. Six protein extraction buffers are available,
all of which are modifications of well-proven protein extraction
buffers designed to produce high spot resolution for 2-D gel analysis.
The buffers are provided in a dry powder formulation, eliminating
carbamylation, a reaction that can occur in solution with urea to alter
protein charge and produce spot artifacts on 2-D spot maps. With
2-D Protein Extraction Buffers, however, buffer can be freshly made
by weighing out the necessary amount and resuspending it in the
included DILUENT.

>> Isolate genomic DNA (gDNA), total RNA, and total denatured
proteins from undivided tissue and cell samples in less than 1 h
>> Directly correlate DNA, RNA, and protein data from the same sample
>> Obtain high yield gDNA, RNA, and protein from precious
limited samples
>> Flexible workflow allows easy isolation of any two or all three analytes
>> Color-coded caps and bottles with matching protocol steps
minimize error
illustra™ triplePrep Kit is designed for rapid simultaneous extraction
and purification of high quality gDNA, total RNA, and total denatured
proteins from animal tissues and mammalian cells. The streamlined
workflow reduces the overall number of steps, resulting in up to 70%
time saving compared to preparing each analyte individually. The highly
purified gDNA, RNA, and protein obtained with the illustra triplePrep Kit
are suitable for downstream genomic and proteomic applications
such as PCR, restriction digestion, sequencing, array CGH, RT-qPCR,
gene expression microarray, SDS-PAGE, Western blotting, 2-D DIGE,
and LCMS. The optimized buffer, columns, and protocol ensure high
recovery of gDNA, RNA, and proteins enabling the use of precious
limited samples such as biopsies, archived tissues, and tumors.

For more information on our range of protein and nucleic acid sample
preparation products, visit www.gelifesciences.com/illustra

Product

Code number

DeCyder 2D Oracle 10gR2, 5-user license

28-9435-88

DeCyder 2D 7.0, additional single-user license

28-9442-77

DeCyder 2D 7.0, single-user trial license

28-9442-79

DeCyder 2D 7.0, single-user license
(upgrade from 6.5 2D)

28-9442-80

DeCyder 2D 7.0, single-user license
upgrade from 6.5

28-9442-81

Image Master 2D Platinum 6.0 DIGE upgrade
to 7.0 DIGE, 1 license

28-9380-80

For more information, visit www.gelifesciences.com/decyder

4

What’s New?

ImageMaster 2D Platinum v7.0

ImageQuant TL
SecurITy Software
Image added to Secure Folder

Working copy created

Analysis performed in
1D gel analysis module

Data stored in Secure Folder
(version number obtained)

No

Analysis complete?
Yes

Results reviewed

No

Results acceptable?
Yes

Data approved

ImageMaster™ 2D Platinum v7.0 is a flexible software package for
visualization and analysis of 2-D gel data, including basic 2-D DIGE
(2-D fluorescence difference gel electrophoresis) and classical 2-D
methodologies. 2-D DIGE is a system that offers significant advantages
over classical 2-D electrophoresis, and ImageMaster 2D Platinum
software uses a patented co-detection algorithm to analyze 2-D DIGE
results. With interactive analysis modes and an improved user interface,
v7.0 offers significant new features for greater convenience and speed,
simplifying even the most complex analysis.
Key features of ImageMaster 2D Platinum v7.0 include:
>> Well-established methods that can be applied to any 2-D experiment

ImageQuant™ TL SecurITy is a supplementary software package
designed to enhance data security in the ImageQuant TL
software. ImageQuant TL SecurITy software allows for greater
control and traceability of 1-D gel electrophoresis data analysis.
ImageQuant TL SecurITy offers added security features
provided by the Version Control Tool and Admin Tool. The Admin
Tool is used to set up user security and restrict user access to
image files, while the Version Control Tool manages multiple
revisions of analyzed 1-D gel images.
ImageQuant TL SecurITy is compatible with all imaging
systems from GE Healthcare.

>> Effective spot detection and matching

ImageQuant TL SecurITy software includes:

>> User-friendly and flexible interface

>> User name and password protection to restrict access to
authorized individuals

For more information and a 14 day free trial of ImageMaster 2D
Platinum, visit www.gelifesciences.com/imp
For more information, visit www.gelifesciences.com/iqtl

5

What’s New?

KappaSelect

Heavy chain
Light chain

Heavy chain
Light chain
VL

FabVH

VL

Fab

VH

VH

V
CL –S–S– CH H

CVHL –S–S

–

VL

Biacore T100
Immunogenicity Package

CL

CH –S–S –S–S–
CL

CL –S–S– CH

–

–S–S–

Fc

–S–S–
–S–S–

IgG

Fc
IgG
CH

VH

CH

VH

CL

Fab

CL

Fab

Binding site for
KappaSelect ligand

VH
Binding site for
KappaSelect ligand
CH

CH

VH

VH

CL

VL

VL
CL

VL

VL

VH

VL

CL

VL
–S–S–

CH

CH

CL

F(ab)2
= Papain digestion

–S–S–

F(ab)2

= Pepsin digestion
—S—S—

= Disulfide bridge
= Carbohydrate

= Papain digestion
= Pepsin digestion
—S—S—

= Disulfide bridge

Antibody structure and binding site for KappaSelect ligand to
= Carbohydrate
Fab fragment.

KappaSelect is an affinity chromatography medium designed
as a Custom Designed Media for the purification of Fab (kappa)
antibody fragments. The ligand binds to the constant region of
the kappa light chain enabling an efficient capture step for
antibody fragments such as Fabs with high purity and yield.

Biacore™ T100 Immunogenicity Package provides tools for the reliable
detection and characterization of anti-drug antibodies (ADAs) during
preclinical and clinical development. In addition to detecting low
affinity antibodies often associated with an early immune response,
the package addresses drug interference by enabling measurement
of ADAs in the presence of excess amounts of drug. New software
tools are also provided for comprehensive characterization of ADAs.
Biacore T100 Immunogenicity Package is sold as an optional module
for Biacore T100.
Biacore T100 Immunogenicity Package allows for:
>> Reliable detection of ADAs: Detect ADAs in the presence of drug
and minimize false negatives by detecting even low affinity ADAs

The Recombinant Protein Purification Handbook: Principles
and Methods has been revised. Main changes include addition
of two chapters, creation of additional selection guides, and
expansion of the desalting chapter to describe new, highthroughput products plus Vivaspin™ sample concentrators.
The first new chapter describes tagging proteins with maltose
binding protein and affinity purification using Dextrin
Sepharose™ High Performance. The second new chapter
describes use of the Strep-tag™ II peptide tag and affinity
purification using StrepTactin Sepharose High Performance.
Both of these chapters plus the expanded desalting chapter
include selection guides and application examples. Updates
were also made in other chapters to include new products
and/or applications.

HiPrep™ Sephacryl™ S-400 HR and S-500 HR columns are the latest
additions to the growing range of columns for chromatographic
purification of molecules by gel filtration (size-exclusion chromatography).
HiPrep Sephacryl HR columns enable a fast and convenient approach
to the purification of large proteins, polysaccharides, and
macromolecules with extended structures. The expanded range of
columns allows purification of molecules with molecular weights as
small as 1 × 103 to macromolecules with molecular weights as large
as 2 × 107.
Two sizes of each are new column are available: 16/60 (column volume
120 ml) for sample volumes of up to 5 ml and 26/60 (column volume 320 ml)
for sample volumes of up to 13 ml.
HiPrep Sephacryl HR columns are prepacked with BioProcess™
validated chromatography media, which are tailored for use in both
laboratory and industrial-scale applications.
In addition, HiPrep Sephacryl HR columns provide:
>> Easy connection to chromatography systems such as the range of
ÄKTAdesign™ systems
>> Reliable and reproducible purifications
>> Convenient scale-up

Ordering information
Product

Code number

HiPrep 16/60 Sephacryl S-400 HR

28-9356-04

HiPrep 26/60 Sephacryl S-400 HR

28-9356-05

HiPrep 16/60 Sephacryl S-500 HR

28-9356-06

HiPrep 26/60 Sephacryl S-500 HR

28-9356-07

For more information, visit
www.gelifesciences.com/protein-purification

GE & Science Prize for Young Life Scientists 2008
Established in 1995, the GE & Science Prize for Young Life Scientists seeks
to bring science to life by recognizing outstanding PhDs from around
the world, and rewarding their research in the field of molecular biology.
Both Science/AAAS and GE Healthcare believe that support of promising
scientists at the beginning of their careers is critical for continued
scientific progress. This year there were over 100 high-caliber entrants,
which made the judging process more challenging than ever. Entrants
submit a 1000 word synopsis of their thesis to the journal Science for
judging by an independent, scientific panel of judges. Each year, one
Grand Prize winner is selected, winning $25 000 in prize money and up
to four regional prizes are also awarded, each receiving $5000. The Grand
Prize winner has his/her essay published in Science and the regional
winners in the online version of Science. The winners attended the
awards ceremony held at Stockholm’s Grand Hotel. In addition, the
prize-winners met the Nobel Laureates in Medicine, Françoise
Barré-Sinousi, Luc Montagnier, and Harald zur Hausen at the Nobel
Q&A session and lunch hosted by Karolinska Institute in Stockholm.
During the two-day event, the award winners also visited the facilities
at GE Healthcare and Uppsala University.

2008 Grand Prize winner
Ethan Clark Garner, the author of the prize-winning essay, “Understanding
a DNA-segregating machine”, was born in Richland, Washington. He
received his BSc in biochemistry from Washington State University,
where he worked with Keith Dunker developing tools to predict disordered
regions within proteins. He conducted his graduate work at the University
of California, San Francisco, where he studied the kinetics and regulation
of prokaryotic polymers with Dyche Mullins. Ethan has since moved to
Boston, where he will be working with Tim Mitchinson, Xiaowei Zhuang,
and Alice Ting on elucidating the process of prokaryotic DNA segregation.

Regional Winners
North America: Xu Tan for his essay “Plant Hormone Auxin Functions as
Novel Molecular Glue”. Dr. Tan spent his first 18 years in Changsha, China.
He became hooked on biology in high school and won a national first
prize in the biology Olympiad. After earning his BSc from the University
of Science and Technology of China in Hefei, he pursued graduate
studies at the University of Washington, Seattle. Under the advice of
Ning Zheng, Dr. Tan did his thesis research on the structural biology of

8

ubiquitin ligases. Looking forward to expanding his research horizons,
he is starting a postdoctoral position with Steve Elledge at Brigham
and Women’s Hospital, Harvard Medical School.
Europe: Sabrina Büttner for her essay “Endonuclease G Regulates
Cellular Fate”. Dr. Büttner was born in Mutlangen, Germany. She studied
biochemistry at the Eberhardt-Karls University, Tübingen, Germany, and
received her diploma with honors in 2004. During her PhD studies,
conducted under the guidance of Frank Madeo at the Institute for
Molecular Biosciences, University of Graz, Austria, she investigated yeast
programmed cell death in the context of aging and oxidative stress,
identifying molecular mechanisms of apoptosis in S. cerevisiae. After
defending her doctoral thesis in 2007, Dr. Büttner continued her research
in the Madeo lab as a postdoctoral fellow, focusing on the further
establishment of yeast as a model for neurodegenerative diseases.
Japan: Kaori Yamada for her essay “Moving PIP3 Regulates Cell Polarity.”
Dr. Yamada grew up in the town of Kinokawa, Japan. She received a BSc
from the University of Tokyo. A strong interest in life science led her to
remain there as a graduate student in Yasuhisa Fukui’s laboratory.
During her postgraduate project, she spent time at the laboratory of
Athar H. Chishti, a collaborator at the University of Illinois, Chicago.
There, Dr. Yamada elucidated how kinesin transports the lipid messenger
PIP3 in neurons. She completed her PhD in January 2007 and is currently
a postdoctoral fellow at the University of Illinois, Chicago.
All other countries: Sarel Fleishman for his essay “Modeling at the Gates
of the Cell.” Dr. Fleishman received an MSc in biochemistry (summa cum
laude) and a PhD (with distinction) from Tel-Aviv University, Israel, where
he studied in the group of Nir Ben-Tal. During his graduate studies, he
investigated the structure, function, and evolution of membrane proteins
associated with hereditary hearing loss and neurodegenerative diseases,
cancer, and bacterial drug resistance. He is currently a Human Frontier
Research Postdoctoral Fellow working on computational design of
protein-based inhibitors toward pathogenic molecules in David Baker’s
laboratory at the University of Washington.
The Grand Prize winning essay, published on page 18 of this issue of
Discovery Matters, is reprinted with permission from AAAS. For the
full text of essays by the regional winners and to apply for this year’s
awards, visit www.sciencemag.org/feature/data/prizes/ge

Starting with cells expressing a histidine-tagged putative transferase
membrane protein, lysis and membrane solubilization was performed
by chemical and freeze-thaw methods in a lysis buffer containing 1%
FC12 detergent. The unclarified lysate was then applied directly to
HisTrap FF crude 1 ml connected to ÄKTAexplorer™ chromatography
system for affinity purification of the tagged protein (Fig 1A). Elution
was performed with buffers containing the detergents, FC12, TCEP,
and DDM.
The eluate from HisTrap FF crude 1 ml step was applied to HiLoad 16/60
Superdex 200 pg. In this step, remaining contaminants as well as
imidazole used for elution from the first chromatography step were
removed using a buffer containing TCEP and DDM. A preliminary size
estimation of the target protein achieved at the same time revealed
that gel filtration effectively removed most contaminants and showed
that no protein aggregates were present in the sample (Fig 1B). To
determine target protein purity, pooled fractions were analyzed by
SDS-PAGE (Fig 1C).
Purification of the membrane protein as described briefly here ensures
a completely pure protein, free from contaminants that would otherwise
interfere with further analysis of the protein by, for example, X-ray
crystallography. This basic method can be modified to allow purification
of other membrane proteins by adjusting detergent composition,
detergent concentration, or pH.

Although this article describes purification using an ÄKTAexplorer
chromatography system, a faster, fully automated approach to the
purification of tagged membrane proteins can be achieved using
ÄKTAxpress™ system.

Fast and efficient purification of histidine-tagged membrane proteins
can be performed by a combination of affinity chromatography on
HisTrap™ FF crude 1 ml column and gel filtration (size-exclusion
chromatography) on HiLoad™ 16/60 Superdex™ 200 pg. This is a fast
and generic purification protocol that does not require optimization.
The imidazole used for elution in the first step is removed during
the gel filtration step together with protein aggregates to give a
highly pure product.

Protein sample preparation

A simple, two-step method for purification of histidine-tagged
membrane proteins

Mr × 103
97

A detailed description of this method can be downloaded from
www.gelifesciences.com, article code number 28-9490-15.

GE Healthcare brings simplicity and versatility to protein extraction
from complex samples prior to downstream protein analysis
methods such as liquid chromatography, 2-D electrophoresis, and 2-D
DIGE. Mammalian Protein Extraction Buffer is designed for efficient
extraction of total soluble protein from both adherent and nonadherent
mammalian cell cultures. Yeast Protein Extraction Buffer Kit is
developed for mild extraction of soluble proteins from yeast cells.
The 2-D Protein Extraction Buffers are six different solubilization
buffers designed to produce high spot resolution for 2-D gel analysis.
The 2-D Protein Extraction Buffer Trial Kit allows you to evaluate
each extraction buffer to find the most suitable buffer for your study.

A)

Buffer kits for extraction of mammalian and
yeast proteins

B)

Mammalian Protein Extraction Buffer and Yeast Protein Extraction Buffer
are based on organic buffering agents, mild nonionic detergents, and a
combination of various salts and agents to gently disrupt the cell wall
and release soluble proteins. Depending on the application, additional
agents such as chelating agents, reducing agents, or protease inhibitors
may be added directly to the extraction buffers before use. Both
buffers are compatible with most downstream applications including
chromatography, electrophoresis, immunoassays, and enzyme assays.
The Yeast Protein Extraction Buffer Kit eliminates the need for glass
beads, a common method of mechanical lysis for yeast cells. A readyto-use Zymolyase™ preparation and Yeast Suspension Buffer are also
provided in the kit. The Yeast Suspension Buffer is used during cell
lysis, together with Zymolyase, an enzyme that digests the cell wall
layer of yeast. After lysis, Yeast Protein Extraction Buffer is added to
the yeast pellet, referred to as spheroplast, to extract biologically
active, soluble protein.

10

2500

Yield (µg)

2000
1500
1000
500
0

1000

800

Yield (µg)

technology CENTRAL

Convenient and efficient methods for cell lysis and
protein extraction

600

400

200

0

High reproducibility using (A) Mammalian Protein Extraction Buffer and
(B) Yeast Protein Extraction Buffer Kit. Total yield for the technical replicates is
shown, as well as the average and standard deviation. In the extraction using
Mammalian Protein Extraction Buffer, 0.3 ml was used to lyse and extract
10 samples containing 1 × 107 CHO cells. One sample is not shown due to
bubbles in the well. In the extraction using Yeast Protein Extraction Buffer Kit,
0.1 ml was used to extract 10 samples containing 50 mg of S. cerevisiae.

2-D Protein Extraction Buffers allow for convenient buffer preparation
and efficient extractions that result in high quality 2-D electrophoresis
results. All six buffers are compatible with 1-D and 2-D electrophoresis
under denaturing conditions and reagents such as protease inhibitor
cocktails may be added to the buffers. Most buffers are compatible
with CyDye™ DIGE Fluors used in 2-D fluorescence difference gel
electrophoresis (2-D DIGE)1.
Tissue sample ≤ 100 mg

The 2-D Protein Extraction Buffer Trial Kit is available to help you
determine the most suitable extraction buffer for your study. Because
some protein spots will be differentially extracted by the buffers, the
most appropriate extraction buffer will depend on the nature of your
sample and the purpose of your study.
1

2-D Protein Extraction Buffer-I is not recommended when using CyDye DIGE Fluor minimal
dyes and Extraction Buffer–III and –IV are not suitable when using CyDye DIGE Fluor Labeling
Kit for Scarce Samples.

Screening for optimal protein extraction using 2-D Protein Extraction Buffers.
In this 2-D protein spot map, spots of protein seen in red were extracted to a
higher degree with Extraction Buffer-II, while spots of protein seen in green
were more highly abundant after extraction with Extraction Buffer-VI.

For more detailed information on products for sample preparation,
visit www.gelifesciences.com/sampleprep
For more information about 2-D gel electrophoresis, visit
www.gelifesciences.com/DIGE

Protein concentration is a crucial step prior to crystallization to obtain
diffraction-quality crystal for X-ray structure determination. In this
study, a two-step purification of histidine-tagged protein upstream
of crystallography is described. Vivaspin™ sample concentrators
were used to concentrate the sample between the two purification
steps, as well as for buffer exchange and sample concentration
prior to crystallization.

Introduction
In structural genomics, a two-step purification, affinity chromatography
followed by gel filtration (size-exclusion chromatography), is often used.
Frequently, sample volume must be reduced before the second
purification step, gel filtration, where size heterogeneities and truncated
forms of the protein can be removed.
For crystallization, a high concentration of protein is required. Vivaspin
sample concentrators are disposable ultrafiltration devices designed
for fast, nondenaturing concentration of biological samples. Vivaspin
can also be used for desalting/buffer exchange.
This study describes a two-step purification of (histidine)6-tagged
MtDXR (MtDXR-[His]6 , Mr 42 000), using affinity chromatography and
gel filtration. Vivaspin was used to concentrate the sample in between
the two purification steps as well as for buffer exchange prior to
crystallization.

to prevent protein precipitation. In the second step, Superdex™ 200 pg,
prepacked in HiLoad™ 16/60 column, was used for gel filtration. The
pooled fractions from gel filtration were buffer exchanged and
concentrated using Vivaspin 20, MWCO 10 000 and further concentration
was performed using Vivaspin 500, MWCO 10 000 intended for smaller
volumes. Protein concentration was determined by measuring UV
absorbance at 280 nm. The fractions were analyzed by SDS-PAGE
under reducing conditions using ExcelGel™ SDS Gradient 8–18. The gel
was stained with Deep Purple™ Total Protein Stain and scanned using
Ettan™ DIGE Imager. Crystallization was performed using the
concentrated protein by the hanging-drop vapor-diffusion technique.

Results
Purification and crystallization of MtDXR-(His)6
In the first purification step, clarified lysate of E.coli expressing the
recombinant protein was purified with HisTrap HP 1 ml (Fig 2). To
remove unwanted E. coli proteins, a linear imidazole gradient was
used for elution. Six milliliter of eluant was concentrated 2.5-fold with
Vivaspin 6, MWCO 10 000 and buffer exchange was performed using
Disposable PD-10 Desalting Column.
Column:
Sample:
Binding buffer:

Fig 1. Workflow for the purification and crystallization of MtDXR-(His)6.

0

5

10

15

20

25

30

35

ml

Fig 2. Affinity purification of MtDXR-(His)6.

Materials and methods
Figure 1 describes the workflow for the purification and crystallization
of MtDXR-(His)6. All chromatography purification was performed using
ÄKTAexplorer™ 100. In the first step, HisTrap™ HP 1 ml column, prepacked
with Ni Sepharose™ High Performance for immobilized metal chelate
chromatography, was used for affinity purification. After the affinity
step, Vivaspin 6, MWCO 10 000 was used to decrease sample volume
and Disposable PD-10 Desalting Column was used for buffer exchange

12

In the second purification step, concentrated eluted pool from
HisTrap HP 1 ml containing MtDXR-(His)6 was loaded on HiLoad 16/60
Superdex 200 pg (Fig 3). The glycerol concentration in the pooled
fractions from gel filtration decreased from 10% to 2.5% using
Vivaspin 20, MWCO 10 000. Furthermore, the protein was
concentrated 20-fold to 3.3 mg/ml using the same concentrator and
Vivaspin 500, MWCO 10 000.

The protein used in this study, MtDXR-(His)6, was generously provided
by L. Henriksson, Dept. of Cell and Molecular Biology, Uppsala University
Biomedical Center, Uppsala, Sweden.

Lo

on

ol

na

Po

tei

rc

Pro

fte

na

Acknowlegements

Mr
97 000
66 000
45 000
30 000
20 100
14 400

Ordering information
Product

28-9322-96

Vivaspin 20, MWCO 10 000*

28-9323-60

Vivaspin 500, MWCO 10 000*

28-9322-25

HisTrap HP, 5 Ă&#x2014; 1 ml

17-5247-01

HiLoad 16/60 Superdex 200 pg column

17-1069-01

Disposable PD-10 Desalting Columns, 30 columns

17-0851-01

ExcelGel SDS Gradient 8â&#x20AC;&#x201C;18, 6 gels

80-1255-53

Deep Purple Total Protein Stain, 5 ml (makes 1 l)
1

2

3

4

5

6

7

Fig 4. SDS-PAGE analysis of MtDXR-(His)6 after purification and concentration.
Electrophoresis was performed under reducing conditions using ExcelGel
SDS Gradient 8-18. The gel was stained with Deep Purple Total Protein Stain
and analyzed using Ettan DIGE Imager.

Code number

Vivaspin 6, MWCO 10 000*

RPN6305

* For all molecular weight cutoffs and the different formats available, see data file 28-9356-53.

For more information about Vivaspin sample concentrators, visit
www.gelifesciences.com/sampleprep

Label-free, real-time protein interaction analysis is an invaluable
tool for the detection and characterization of unwanted immune
responses. Screening for anti-drug antibodies with Biacore™
instruments has the advantage of detecting early immune responses
characterized by low affinity antibodies that interact with the drug
with rapid kinetics. These immune responses can be clinically
significant, but are easily missed by alternative endpoint assay-based
techniques due to losses during washing procedures etc. The new
Biacore T100 Immunogenicity Package from GE Healthcare offers
dedicated support for immunogenicity testing by providing tools for
screening, confirmation, and characterization of immune responses.
It also addresses drug interference by enabling the measurement
of anti-drug antibodies in the presence of excess amounts of drug.

higher sensitivity, the importance of detecting both early and late
immune responses resulted in Boehringer Ingelheim implementing
Biacore as their choice of immunogenicity screening platform.

Detection of anti-drug antibodies in the
presence of drug
Drug present in serum samples can bind to ADAs and interfere with
the detection step, thereby generating false negatives. This is a
general problem for immunogenicity assays, and in Biacore assays,
drug interference would prevent ADAs from binding to the surface of
the sensor chip (Fig 2).
Increasing concentration of drug

Introduction
Most biopharmaceuticals elicit some level of antibody (Ab) response
against the drug, which in some cases can lead to serious adverse
effects and/or loss of efficacy. Immunogenicity is therefore an
important factor that must be considered in the development of new
biotherapeutics, particularly due to extensive regulatory demands.
The market for biotherapeutics is a rapidly growing area, and
consequently, there is an increasing need to study immune responses
in preclinical and clinical development.

Reliable detection of anti-drug antibodies
Detection and characterization of unwanted immune responses is an
important aspect of drug safety studies. Biacore T100 Immunogenicity
Package can detect these immune responses that may be missed by
alternative endpoint assay-based techniques, due to losses during
washing procedures etc. Screening for anti-drug antibodies (ADAs)
with Biacore instruments has the advantage of detecting early immune
responses characterized by low affinity antibodies that interact with
the drug with rapid kinetics.

Fig 2. Excess amount of drug in samples may inhibit ADAs from binding to
the sensor chip surface.

Biacore T100 Immunogenicity Package addresses the issue of drug
interference by enabling measurement of ADAs in the presence of
excess amounts of drug. Samples are acidified to dissociate drugantibody complexes, and then neutralized just before measurement
to prevent complexes from reforming (Fig 3). This enables a sensitivity
of < 0.5 μg/ml Ab in the presence of 100-fold molar excess of drug.
Acidification

Neutralization

Type II immune response
“persistent”

1400
1200

30000

1000

20000

800
600

10000

Cut-off

400
200

ELISA (ng equiv./ml)

B)

ELISA (ng equiv./ml)

A)

Type I immune response
“transient”

1400
1200

30000

1000

20000

800
600

10000

Cut-off

400

Biacore (ng equiv./ml)

Fig 3. Acidification and neutralization allows the Biacore assay to be performed
accurately in the presence of excess amount of drug. Samples are acidified
to dissociate drug-antibody complexes, and then neutralized just before
measurement to prevent complexes from reforming.

Biacore (ng equiv./ml)

In Figure 1, Boehringer Ingelheim compared a Biacore assay with a
bridging ELISA in a phase I, multidose clinical study for a therapeutic
humanized Ab. Figure 1A shows that Biacore T100 was able to detect
ADAs much earlier than the ELISA in subjects displaying a type II or
“persistent” immune response. In subjects displaying a type I or “transient”
immune response, the Biacore assay detected several positives while
the ELISA did not (Fig 1B). Although the ELISA assay formally had a

In a study using Rituximab, different concentrations of anti-Rituximab
Abs were mixed with increasing amounts of drug (Fig 4). The detection
of ADAs was inhibited as the drug concentration increased. Using the
acidification and neutralization steps, the ADA response could be
measured even in the presence of an excess amount of drug.

Anti-Rituximab, 0.5 µg/ml in serum

Anti-Rituximab, 0.5 µg/ml in
serum, acidified and neutralized

0

0.25

0.5

5

Fig 5. Rat serum bound to the sensor surface was challenged sequentially
with isotype-specific reagents. The binding responses indicate the presence
and relative proportions of different isotypes in the sample Ab population,
and in this case, the isotype was mainly IgG2a.

50

Rituximab (µg/ml)

Fig 4. Anti-Rituximab Abs (0.5 μg/ml) were mixed with increasing amounts of
Rituximab in negative human serum. Due to drug interference, the measured
ADA response decreased as the Rituximab concentration increased (curve A).
When anti-Rituximab Abs at 0.5 μg/ml were mixed with drug and the samples
acidified and neutralized prior to measurement, the ADA response was
completely recovered (curve B).

Comprehensive characterization of ADAs
Biacore T100 Immunogenicity Package offers dedicated software
tools for comprehensive characterization of immune responses.
To characterize Ab class and subclass using Biacore systems, the
antibodies in the sample are bound to the sensor surface, challenged
with a series of isotype-specific reagents (typically antibodies), and
the binding responses indicate the presence and relative proportions
of the different Ab isotypes present. In Figure 5, rat serum was bound
to the sensor surface and sequential injection of different anti-isotype
reagents showed that the sample Ab population consisted mainly of
IgG2a. With Biacore systems this type of assay is simplified and data is
generated in real-time, whereas ELISA usually requires one microplate
for each Ab isotype tested, making it more cumbersome.
In addition, Biacore systems enable monitoring of Ab maturation via
assessment of binding stability. The Ab population in positive samples
can be characterized in terms of the binding stability to the drug on
the sensor surface. ADAs usually represent a heterogeneous population
with a spectrum of different properties. Usually, using a simple exponential
decay to study dissociation does not properly fit the data, in part
because of the heterogeneous Ab population, but also because Abs
are bi- or multivalent, and avidity influences binding stability. Software
tools in the Biacore T100 Immunogenicity Package can be used to
characterize the immune response in terms of Ab fractions or populations
with rapid and slow dissociation rates.

Conclusions
Label-free, real-time Biacore assays give vital information that cannot
be provided by alternative technologies, by enabling detection and
characterization of low affinity Abs with rapid off-rates. Biacore T100
Immunogenicity Package provides dedicated tools for reliable detection
and characterization of ADAs during preclinical and clinical development.
In addition to detecting low affinity Abs often associated with an early
immune response, the package addresses drug interference by enabling
measurement of ADAs in the presence of excess amounts of drug.
Software tools are also provided for comprehensive characterization
of ADAs. Biacore T100 Immunogenicity Package, available as an optional
module for Biacore T100, offers one platform that can be used throughout
the immunogenicity workflow from screening to characterization of
immune responses.

Acknowledgements
We would like to thank Dr. U. Kunz (Boehringer Ingelheim Pharma
GmbH & Co. KG, Germany) for kindly contributing data, and
Dr. D. Mytych (Amgen, Inc., USA) whose group we collaborated with
during the project.

illustra™ triplePrep Kit enables the isolation of high quality genomic
DNA, total RNA, and total denatured protein from a single undivided
sample. Comparative analysis revealed higher yields of equal or higher
quality than those obtained from the same input sample amounts
using DNeasy™ and RNeasy™ kits (Qiagen™) and 2-D fluorescence
difference gel electrophoresis (2-D DIGE).
Until recently, researchers in fields such as functional genomics, molecular
genetics, and biomarker studies used three separate kits to isolate
DNA, RNA, and proteins for use as probes and targets in downstream
applications. However, the use of divided samples could potentially
skew results due to heterogeneity between different cell and tissue
samples. The demand for good correlation between transcript (gene)
expression, protein expression, copy number variation, and SNP
detection has resulted in the development of illustra triplePrep Kit.
This new kit enables the isolation of high quality genomic DNA (gDNA),
RNA, and proteins from a single undivided source of tissue or cells in
less than one hour.
We tested the performance of illustra triplePrep Kit by using animal
tissues from different organs (e.g., liver, kidney, spleen, brain, lung, and
intestine) or cultured mammalian cells (e.g., HeLa, NIH-3T3, CHO-K1,
and HEK-293). Analysis of data related to yields and purity showed
superior performance of illustra triplePrep Kit compared to the DNeasy
Blood and Tissue Kit and RNeasy Mini Kit from Qiagen and 2-D
fluorescence difference gel electrophoresis (2D-DIGE), the reference
method for protein isolation (1).

The procedure used for purification of DNA, RNA, and protein is described
in Figure 1. Tissue or cells were lyzed in lysis buffer, loaded onto a DNA mini
column, and gDNA eluted with elution buffer. Acetone was added to
flowthrough containing RNA and protein and loaded onto an RNA mini
column. Following RNA binding to the second column, DNase was used
to remove any remaining gDNA contamination. The flowthrough from
the RNA column containing only proteins was isolated by precipitation.
Purity of nucleic acids was measured by NanoVue™ spectrophotometer
and quality was measured using Agilent™ Bioanalyzer. Total protein
yield was compared with 2-D DIGE using 2-D Quant Kit and the quality
of total proteins isolated was tested by Western blotting and LC-MS.

Results
Genomic DNA
gDNA was isolated from 10 mg of rat liver tissue using the illustra
triplePrep Kit or the DNeasy Kit. Yield, purity, and quality were compared
for both samples (Fig 2). gDNA was used to amplify a 1.5 kb fragment by
PCR. The resulting fragment was then sequenced to compare performance
in downstream applications. Results showed higher than average yields
with illustra triplePrep Kit (15.4 µg ± 0.60) compared to DNeasy Blood
and Tissue Kit (9.8 µg, ± 2.10) using the same amount of input sample.
gDNA isolated from both kits produced similar purities as determined
by A260/A280 optical density ratios (1.90 ± 0.02). The sequenced PCR
fragment generated from illustra triplePrep Kit showed higher Phred
illustra triplePrep Kit

DNeasy Kit

Materials and methods
DNA, total RNA, and total protein were extracted from 10 mg of rat liver
tissue or 1 × 106 HeLa cells using the illustra triplePrep Kit. The performance
of this kit was compared against kits used to isolate gDNA (DNeasy
Blood and Tissue Kit, Qiagen), RNA (RNeasy Mini Kit, Qiagen), and protein
(2-D DIGE) individually. Comparative analysis was based on sample
input amount, yield, purity, speed, and ease-of-use following the
manufacturers’ recommended protocols.
DNA purification

RNA purification

Protein isolation

1. Sample homogenization
and lysis

2. DNA binding

20 scores (744 bp) compared to DNeasy Blood and Tissue Kit (723 bp)
indicating the suitability of gDNA in downstream applications such as
PCR and sequencing.

Total RNA
Total RNA was isolated from 10 mg of rat liver tissue using illustra
triplePrep Kit or RNeasy Mini Kit. Yield, purity, and quality were compared
for both samples (Fig 3). Both kits yielded high quality total RNA (Fig 3A).
The total RNA samples were used to amplify high-, medium-, and lowexpressed 18S ribosomal RNA, cytochrome P450, and c-fos gene,
respectively by real-time quantitative PCR (RT-qPCR). Similar slopes and
CT values were obtained in all three cases, indicating similar performance
of the kits in downstream applications (Fig 3B). Total RNA isolated from
both kits produced similar quantities and purities as determined by
A260/A280 ratios (data not shown).

Proteins were isolated from the second flowthrough after isolating
gDNA and total RNA following the illustra triplePrep protocol. Yield was
similar for the both illustra triplePrep Kit and 2D-DIGE reference method
(Fig 4A). Western blotting using appropriate antibodies was used to
detect β-actin or GAPDH proteins (Fig 4B). The peptides were separated
by nanoRPC on Ettan™ MDLC coupled to a Finnigan LTQ™ Linear Ion
Trap mass spectrometer (Thermo Fisher Scientific Inc.). The LC-MS
data was evaluated using DeCyder™ MS Differential Analysis Software.
Protein samples from the illustra triplePrep Kit extraction were also
analyzed by LC-MS for peptide profiling and showed average
representation of 3115 ± 176 peptides (Fig 4C).

Understanding a minimal DNA-segregating machine
Reconstitution of a plasmid spindle shows how three components together
accomplish the task of DNA segregation
E. C. Garner
Systems Biology, Harvard Medical School, Boston, MA, USA
A current challenge in biology is to bridge the gap between the parts
and the whole, to reconcile the biochemical properties of individual
proteins with the emergent behaviors of multipart molecular machines.
In theory, if every kinetic rate constant for every interaction were
measured, we could gain a complete mechanistic understanding of a
biological process. Because the number of required measurements
scales with the number of components, most complex biological
systems present difficult challenges for this approach. However, when
quantitative insight is matched with the proper system, the goal of
elucidating the biochemical basis of emergent behaviors can be realized.
For example, to gain a molecular-level understanding of DNA segregation,
a fundamental, mesoscale process, I turned to a system that has been
forced to be minimal and self-contained during its evolution: low-copy
bacterial plasmids. To maintain their inheritance against the fitness
costs imposed by their extra metabolic burden, these exogenous elements
have evolved minimal, self-contained, DNA segregation machines.
At a minimum, a DNA-segregating system needs to accomplish three
tasks. First, it must count the copies of DNA to know when to initiate
segregation. Second, it must exert directional force to propel these
DNA copies away from each other. Third, this system must be spatially
aware of the cellular geometry so that the DNA is propelled toward
each eventual daughter cell. For the Escherichia coli R1 drug resistance
plasmid, all of these tasks are conferred by the par operon, which
constructs a mitotic spindle out of only three components (1). The
centromeric sequence parC contains 10 repeats that are bound by the
adapter protein ParR. This ParR/parC complex interacts with ParM, a
distant actin homolog that polymerizes into helical filaments (2). In my
thesis work, I determined how these three components interact to
accomplish the systems-level task of DNA segregation. To elucidate
this mechanism of plasmid segregation, I needed to understand the
nature of ParM polymerization, how the ParR/parC complex affected
ParM filaments, and finally, how these three components interact to
push the plasmids to the poles of the cell.
First, I conducted a complete characterization of ParM assembly
dynamics, measuring all available rate constants for this polymer (3).
Although ParM is a structural homolog of eukaryotic actin, I found
three distinct differences between these polymers. First, ParM nucleates
filaments at a rate 300 times as fast as actin. Second, unlike any other
previously observed polymer, ParM shows no polarity as it grows at equal
rates at each end of the filament. The most striking difference between
ParM and actin is that ParM exhibits dynamic instability, the stochastic
switching between states of growth and rapid disassembly (4).
Previously, this behavior had only been observed for eukaryotic
microtubules. ParMâ&#x20AC;&#x2122;s dynamic instability is driven by ATP hydrolysis, as
ADP-bound ParM filaments display a much higher dissociation rate
than the ATP-bound filaments.
The combination of dynamic instability and rapid nucleation causes
solutions of ParM to consist of a population of short, dynamic filaments
that nucleate and turn over throughout the volume (Fig 1). This unstable,

18

transient nature appears at odds with the formation of a rigid forcegenerating mitotic spindle, suggesting that the ParR/parC complex must
alter ParM filament dynamics. I tested this hypothesis by reconstituting
the par system from purified components (5), combining parC-conjugated
beads with ParR and fluorescently labeled ParM. Isolated parC beads
displayed short, dynamic asters of ParM emanating from their surface,
as if they were searching the surrounding volume. When two parC
beads came into close contact, stable bundles of filaments formed
between the beads. These filaments then elongated at a constant
rate, pushing the beads in opposite directions.

Fig 1. Dynamic instability and bipolar stabilization drive plasmid segregation.
The ParR/parC complex can capture cytoplasmic ParM filaments. Filament
ends bound by ParR/parC are stabilized (blue) while unbound filaments
(red) and destined to undergo catastrophe. A productive spindle is formed
when both ends are bound by ParR/parC. Turnover of the unattached,
background filaments provides the energy differential (arrows) to power
spindle elongation.

Creating fiducial marks on these filaments demonstrated that they
elongate at equal rates at each bead surface, indicating that new
monomers are added into the spindle through a process of insertional
polymerization at the ParR/parC complex. This was the first in vitro
reconstitution of a DNA-segregating system from purified components.
The fact that we observed long, stable ParM filaments only between
pairs of beads indicated that the filaments are stabilized against
catastrophic disassembly when bound at both ends by ParR/parC.
When these spindles were severed with laser irradiation, these filaments
would rapidly depolymerize. This bipolar stabilization provides an
intrinsic counting mechanism, as productive and sustained filament
elongation only occurs between parC pairs.
I then tested whether these spindles could locate the ends of a volume
by confining them in microfabricated channels of various shapes.
These spindles aligned with and elongated along the long axis of these
spaces, indicating that this simple system is sufficient to find the long
axis of a cell.

GE & Science Prize 2008

In addition to demonstrating that these three components are
necessary and sufficient to generate the emergent behaviors required
for DNA segregation—counting, force generation, and spatial
awareness—this work also elucidated the biophysical relationship
between dynamic instability and force generation. By conducting
spindle assembly assays at varying concentrations of ParM, I found
that filaments bound at each end by ParR/parC behave as if the entire
filament is composed of the higher-affinity, ATP-bound polymer. In essence,
this stabilizes the bound filaments to a lower energetic level than the
unbound filaments that turn over in solution (see Fig 1). This indicates
that the dynamic instability of the unattached filaments provides the
monomer excess that powers the elongation of the stabilized ParR/parC
attached filaments.
These studies provide a framework for understanding the essential
principles of DNA segregation. Furthermore, they demonstrate that
biology can solve complex tasks with a surprisingly small number of
components. This minimal solution to DNA segregation is not an
isolated case; many low-copy plasmids have independently converged
on three-component systems by co-opting a variety of different
polymers from their hosts (6–9). Kinetic dissection of a range of these
self-contained mitotic machines may uncover the differing solutions
that biology has evolved to ensure genetic inheritance and thus
broaden our understanding of this fundamental biological process.

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